1. Field of the Invention
The present invention is directed to a method and device for collection of airborne particles and their concentration in very small amounts of liquid (μL) for further analysis and investigation. The method can be applied for the sampling and/or collection of airborne particles of non-biological as well as of biological origin, such as bacteria, fungi and their mixtures. Ability of the proposed method to concentrate the collected biological airborne particles into extremely small amounts of liquid increases sensitivity of this method and device and makes them especially applicable for biodefense purposes.
2. Description of Related Art
Exposure to airborne biological agents, especially pathogenic or allergenic microorganisms, may cause a wide range of respiratory and other health disorders in occupational and general populations. Moreover, health-care professionals increasingly recognize bioaerosols as a cause of preventable airborne infections and hypersensitivity diseases (WHO, 1990). Although relatively little data exist on the presence of cells or cell material of fungi and bacteria in fine particle samples (Womiloju et al., 2003), some studies indicate that perhaps as many as 10% of urban and rural fine aerosols are biological in nature (Monn, 2001), which stresses the need to develop advanced tools needed to assess and control our exposure to airborne microorganisms (bioaerosols) indoors and outdoors, and to protect the populations and resources potentially exposed to airborne microbial agents. It has been concluded that many bioaerosol species that may cause health effects are currently not yet identified and more research is needed to establish better tools for assessing exposure to biological aerosols (Douwes et al., 2003). In addition, improved exposure assessment and protection of populations and resources at risk from biowarfare agents requires advanced air sampling devices that feature high collection efficiency and can detect low agent concentrations.
Currently, bioaerosols are commonly collected using techniques that require active sampling, such as impaction, impingement or deposition on filters. Recently, there has been an increased interest in collection of microorganisms using electrostatic precipitation due to its lower power requirements compared to inertial techniques while still allowing efficient particle removal from the air. In electrostatic precipitators, airborne particles are electrically charged and then removed from the air stream by electrostatic field. Removal of non-biological aerosol particles by electrostatic precipitators has been widely studied from the theoretical and practical points of view (Rose and Wood, 1956; Lu and Hungsung, 1998; Zhuang et al., 2000), owing to its widespread practical applications. These devices provide efficient particle capture while causing minimal impedance to the gas flow.
An investigation and a successful practical application of electrostatic precipitation for collection and enumeration of viable airborne microorganisms have also been described. The studies showed that aerosolized Pseudomonas fluorescens bacteria can carry up to 13,000 elementary charges (Mainelis, 2001) and that they can be effectively collected by an electrostatic technique (Mainelis et al., 2002a; Mainelis et al., 2002c). It was also found that electrostatic fields are unlikely to damage the organisms passing through an electrostatic collector or those microorganisms already deposited on an agar medium (Yao et al., 2005). Based on these observations, an electrostatic precipitator that utilizes natural microorganism charge for their collection was developed and tested (Yao and Mainelis, 2006b). Investigations in indoor and outdoor environments of said sampler showed that it recovered from 5 to 10 times higher concentrations of culturable microorganisms compared to a traditional impactor.
A method used to analyze biological and non-biological particles collected by various samplers depends on the sampling medium. Liquid is often a preferred sampling medium because of its versatility. Biological particles in liquid samples can be analyzed by numerous techniques, including, but not limited to, microscopy, culturing techniques, and various microbiological techniques, such as polymerase chain reaction (PCR), enzyme-linked immuno assays (ELISA) and similar. Performance of a liquid-based aerosol collector to detect the presence of airborne particles is determined not only by its collection efficiency, but also by its concentration rate. High concentration rates reduce the sampling time needed to detect airborne particles and enable detection of lower particle concentrations (Haglund, 2003). The concentration rate is defined as the rate with which particles present in an air volume are concentrated in a liquid volume per time period:
                                                                        Concentration                ⁢                                                                  ⁢                rate                            ,                                                                    R                    C                                    ⁢                                                                          ⁢                                      (                                          t                                              -                        1                                                              )                                                  =                                ⁢                                                      Airborne                    ⁢                                                                                  ⁢                    particle                    ⁢                                                                                  ⁢                    concentration                    ⁢                                                                                  ⁢                                          (                                              L                                                  -                          1                                                                    )                                                                            Particle                    ⁢                                                                                  ⁢                    concentration                    ⁢                                                                                  ⁢                    in                    ⁢                                                                                  ⁢                    liquid                    ⁢                                                                                  ⁢                                          (                                              L                                                  -                          1                                                                    )                                                                                                                                                              =                                ⁢                                                                            Q                      ⁡                                              (                                                  L                          /                          min                                                )                                                                                    v                      ⁡                                              (                        L                        )                                                                              ⁢                  η                                            ,                                                          (        1        )            where Q is the sampling flow rate, v is the sample volume and η-collection efficiency. Traditional liquid samplers operate at flow rates up to 20 L/min and feature low sample concentration rates, e.g. up to 2,500 for BioSampler (SKC, Inc., Eighty Four, Pa.) operating at 12.5 L/min and sampling into 5 mL of liquid. Since the anthrax attacks of 2001, several new samplers have been developed for collecting airborne particles into liquid. Among those, InnovaTek, Inc. (Richland, Wash.) introduced the BioGuardian Air Sampler which operates from 100 to 1000 L/min and collects sample into 10-15 mL of liquid. The SpinCon air sampler by Evogen, Inc. (Kansas City, Mo.) samples at 450 L/min and concentrates sample into 10 mL of liquid. The BioCapture 650 (MesoSystems Technology, Inc., Albuquerque, N. Mex.) is a portable sampler that achieves a sampling flow rate of 200 L/min and collects particles into 2-5 mL of liquid. The concentration rates for these samplers are in the order of tens of thousands. A new wetted-wall bioaerosol cyclone developed at the Texas A&M University has concentration rates of approximately 5×104/min for bacteria-sized particles (Seo, 2007). The Lawrence Livermore National Laboratory (Livermore, Calif.) has developed a stationary Autonomous Pathogen Detection System (APDS) that combines a virtual impactor and a wetted-wall cyclone and is capable of continuous and fully autonomous monitoring for multiple biowarfare organisms (McBride et al., 2003; Hindson et al., 2004; Hindson et al., 2005a; Hindson et al., 2005b). The APDS operates at collection flow rates up to 3750 L/min and can achieve concentration rates as high as 7.5×105/min when collecting 3 tan polystyrene latex (PSL) particles into 4 mL of liquid (Mainelis et al., 2005). However, the size of the system, its power and cost requirements are not conducive for its mass deployment.
Given the low power consumption of electrostatic precipitators, several models have been developed to collect particles into liquid. Particles collected electrostatically into liquid can be easily transferred into various analytical devices, such as “laboratories-on-a-chip” which is especially advantageous for the detection and identification of biological agents. U.S. Pat. No. 6,955,075 describes a briefcase-sized electrostatic precipitator that samples at air flow rate of 300 L/min (Carlson, 2005). The particles are electrically charged, deposited onto a vertically tubular collection electrode and continuously washed-off by recirculating liquid. The amount of liquid is not specified, although a presentation by the same authors indicated 20 mL (Carlson, 2004). The U.S. Pat. No. 7,428,848 describes a high throughput electrostatic collector (Pant et al., 2008) where particles are deposited onto a horizontally-oriented collection electrode and are washed off into liquid. One particular embodiment collects particles at a flow rate of 60 L/min into 10 mL of liquid. These samplers mentioned above have maximum concentration rates of about 6,000-15,000/min. Another wet electrostatic collector is described in the U.S. patent application Ser. No. 11/473,748 (Zaromb and Martell, 2007). The device achieves collection efficiency of 94% when collecting 1 μm PSL beads at a flow rate of 510 L/min. The particles collected on the tubular collection electrode are washed of with 3-10 mL of liquid injected at intervals of 5-20 seconds. In one of the described experiments, particles were accumulated in 60 mL of liquid.
Thus, the concentration rates of majority of aerosol/bioaerosol samplers, especially the compact ones, are still in the order of tens of thousands even when assuming 100% collection efficiency. Since the increase in a sampler's concentration rate improves its capabilities to detect a particular biological agent or a pollutant it is important to develop samplers that feature high concentration rates. Compared to inertia-based techniques, electrostatic precipitators require less energy, and thus development of electrostatic samplers capable of high concentration rates is especially beneficial. As could be seen from Eq. 1, the concentration rate could be improved either by increasing the sampling flow rate or by decreasing the sample volume. The electrostatic collectors described in prior art feature sample volumes from 10 to 60 mL (milliliters). In this patent application a method and a device are presented where the sample volume is reduced by three orders (1000×) of magnitude, i.e., to 5-60 μL (microliter). This reduction is critical because modern sample analysis tools use only a fraction of the sample (microliter amounts) for the actual analysis of the sample (Hindson et al., 2005a) and thus the entire sample could be analyzed thus increasing sensitivity of detection.
In this application I present a method and a device, where a combination of electrostatic collection mechanism and a collection surface coated with superhydrophobic material (water contact angle >150°) allows effective collection of particles and, more importantly, allows achieving very high concentration rates. The principle of the method and its ability to achieve concentration rates exceeding 1×106/min distinguish this method and device from prior art.
In nature, superhydrophobic surface properties allow for certain plants (“Lotus leaf” type) to be cleaned from dust pollution by a simple rain shower (Barthlott and Neinhuis, 1997). Superhydrophobic nature (high contact angle) of the leaf's surface makes water droplets form spheres with very little adhesion to the surface and the droplets roll off very easily even at small inclinations under the force of gravity. Microscopic examination of such surfaces revealed the presence of micro-structured surface as well as coating by water-repellent crystals (Ma and Hill, 2006). When a water droplet rolls over a particle deposited on such a surface, the particle is wetted, adheres to the droplet and is removed from the surface. Thus, this “self-cleaning” property of superhydrophobic surface is combined with electrostatic collection mechanism into a new method and a device. Here, the airborne particles are electrostatically deposited onto a superhydrophobic surface from where they are removed and collected by small rolling water droplets (from 5 to 60 μL) for subsequent analysis. In this Electrostatic Precipitator with Superhydrophobic Surface (EPSS) the airborne particles are collected for a desired period of time and the deposited particles are then removed at the end of the sampling period by one small liquid droplet thus accruing all the collected particles in one droplet. Since the deposit is suspended in liquid, the presence of biological/non-biological particles or chemical substances in the sample can be determined by multiple analytical techniques. For biological particles this includes, but is not limited to, the traditional culturing and microscopy techniques as well as the modern molecular analysis tools, such as PCR or ELISA.